兰州大学机构库 >口腔医学院
Sestrin2通过调节mTOR和MAPK通路对口腔鳞状细胞癌的作用及机制研究
Alternative TitleThe effect and mechanism of Sestrin2 on oral squamous cell carcinma through mTOR and MAPK pathway regulation
刘泽琳
Subtype硕士
Thesis Advisor刘斌
2023-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline口腔临床医学
KeywordSesn2 Sesn2 口腔鳞状细胞癌 Oral Squamous Cell Carcinoma mTOR mTOR MAPK MAPK 肿瘤发生 Tumorigenesis
Abstract

目的:本研究旨在探讨 Sestrin2(Sesn2)对口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)细胞系的增殖、迁移、侵袭和凋亡的生物学行为影响及基 于 PI3K/AKT/mTOR 和 MAPK 通路的机制初步探讨。 方法:首先从癌症基因组图谱(The Cancer Genome Atlas,TCGA)中收集了 Sesn2 在正常组织和口腔鳞状细胞癌项目中相关的 RNA-seq 数据,进行配对样本 和非配对样本差异分析;同时,从 HPA 数据库(The Human Protein Atlas, HPA) 中获取免疫组织化学染色图像,共同评价 Sesn2 在 OSCC 中的表达和潜在作用; 采用小干扰 RNA(small interfering RNA, siRNA)和慢病毒转染技术,分别敲减 和过表达 Sesn2 基因,随后通过 CCK-8 法和 Brdu 法来检测 Sesn2 对 CAL27 和 TSCCA 细胞系在细胞增殖与毒性以及增殖活力方面的影响;采用流式细胞术检 测 Sesn2 对 CAL27 和 TSCCA 细胞周期分布的影响;采用 Trans-well 法和细胞划 痕法来反映 Sesn2 对 CAL27 和 TSCCA 细胞系体外迁移和侵袭能力的影响;通 过 Hoechst 33342 染色和 Caspase 3 酶活性检测实验,观察 CAL27 和 TSCCA 细 胞的核形态和酶活性变化从而评价 Sesn2 对两种细胞系体外凋亡的影响;最后, 采用 Western Blot 法来检测 PI3K/AKT/mTOR 和 MAPK 通路中关键分子蛋白的 表达水平。 结果: (1)根据 RNA-seq 数据的分析数据结果显示,对于非配对样本差异分析中, Sesn2 在 OSCC 中的表达量比正常组织高约 10.6%(P<0.001),在配对样本差异 分析中,Sesn2 在 OSCC 中的表达量比正常组织高约 12.5%(P<0.01);同时, HPA 数据库获取的免疫组织化学染色图像结果显示 Sesn2 的表达水平在头颈部 鳞状细胞癌中更高。 (2)根据 CCK-8 和 Brdu 检测的结果显示,敲减 Sesn2 可抑制 CAL27 和 TSCCA 细胞体外的增殖;并使得细胞周期中 G0/G1 的比例下降,S 期无发生明 显变化,G2/M 期升高;同时 Trans-well 法和细胞划痕实验显示,Sesn2 表达抑制 可显著降低两种细胞系的迁移和侵袭能力;此外,CAL27 和 TSCCA 细胞的凋亡 II 小体和 Caspase 3 酶活性增加,细胞凋亡增加;过表达 Sesn2 后 CAL27 细胞的各 种生物学行为与之相反。 (3)敲减 Sesn2 后,PI3K、P-AKT、P-ERK、P-P38、mTOR、MMP-2、MMP9 和 MMP-7 的蛋白表达水平均下降,Caspase 3 蛋白的表达含量增加,而过表达 Sesn2 后与之相反;然而,过表达和敲减 Sesn2 后,BAX/BCL-2 都没有明显变化。 结论: Sesn2 可能通过 PI3K/AKT/mTOR 和 MAPK 途径在口腔鳞状细胞癌的发生、 发展起着促进作用。

Other Abstract

Objective: The purpose of this study is to investigate the biological effects of Sestrin2 (Sesn2) on the proliferation, migration, invasion, and apoptosis of oral squamous cell carcinoma (OSCC) cell lines while preliminarily exploring the mechanisms based on the PI3K/AKT/mTOR and MAPK pathways. Methods: Firstly, RNA-seq-related data to Sesn2 in normal tissues and oral squamous cell carcinoma projects were collected from The Cancer Genome Atlas (TCGA), differential analysis was performed on paired and unpaired samples. Meanwhile, immunohistochemical staining images were obtained from The Human Protein Atlas (HPA) database to evaluate the expression and potential role of Sesn2 in OSCC. Then, small interfering RNA (siRNA) and lentivirus transfection techniques were used to knockdown and overexpress the Sesn2 gene, respectively. Subsequently, the effects of Sesn2 on cell proliferation, toxicity, and viability were measured by CCK8 and Brdu assay in the CAL27 and TSCCA cell lines. The effects of Sesn2 on the cell cycle distribution of CAL27 and TSCCA cells were detected through a flow cytometry. The Trans-well and cell scratch assays were used to reflect the effects of Sesn2 on the in vitro migration and invasion abilities of CAL27 and TSCCA cell lines. Hoechst 33342 staining and Caspase 3 enzyme activity detection were used to evaluate the effects of Sesn2 on the in vitro apoptosis of both cell lines by observing changes in nuclear morphology and enzyme activity. Finally, the expression levels of key molecular proteins in the PI3K/AKT/mTOR and MAPK pathways were detected by Western Blot. Results: (1) According to the analysis of RNA-seq data, the expression of Sesn2 in OSCC was 10.6% higher than that in normal tissues in the analysis of unpaired samples (P<0.001). In the difference analysis of paired samples, the expression of Sesn2 in OSCC was 12.5% higher than that in normal tissues (P<0.01). At the same time, the IV results of immunohistochemical staining images obtained from HPA database showed that the expression level of Sesn2 was higher in head and neck squamous cell carcinoma. (2) According to the results of CCK-8 and Brdu detection, the knockdown of Sesn2 inhibited the proliferation of CAL27 and TSCCA cells in vitro, which caused that the proportion of G0/G1 in the cycle decreased while that of G2/M phase increased without significant change in S phase. At the same time, Trans-well assay and Cell Scratch assay showed that the inhibition of Sesn2 expression significantly reduced the migration and invasion ability of both cell lines. In addition, the apoptotic bodies and Caspase 3 enzyme activity of CAL27 and TSCCA cells increased with the increase of apoptosis. However, the biological behaviors of CAL27 cells after overexpression of Sesn2 were on the contrary. (3) The protein expression levels of PI3K, P-AKT, P-ERK, P-P38, mTOR, MMP2, MMP9 and MMP-7 decreased while the expression of Caspase 3 protein increased after Sesn2 being knockdown. However, it is the opposite while there was no significant change in BAX/BCL-2 after overexpression and knockdown of Sesn2. Conclusion: Sesn2 may play a stimulative role in the development and progression of oral squamous cell carcinoma through PI3K/AKT/mTOR and MAPK pathways.

Subject Area口腔肿瘤学
MOST Discipline Catalogue医学 - 口腔医学 - 口腔临床医学
URL查看原文
Language中文
Other Code262010_220200926840
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537141
Collection口腔医学院
Affiliation
兰州大学口腔医学院
Recommended Citation
GB/T 7714
刘泽琳. Sestrin2通过调节mTOR和MAPK通路对口腔鳞状细胞癌的作用及机制研究[D]. 兰州. 兰州大学,2023.
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